Abstract. Existing antiretroviral drugs do not clear HIV-1 latent reservoirs, underscoring the urgent need for new therapeutic strategies. The HIV-1 Nef accessory factor is an attractive target for drug development because of its critical roles in the HIV-1 life cycle and immune system escape. Our group has discovered novel small molecules that bind directly to Nef and block many of its functions, including enhancement of viral infectivity and replication in donor PBMCs. Importantly, our Nef inhibitors rescue cell-surface MHC-I expression in latently in- fected, patient-derived CD4 T-cells, enabling recognition and killing by autologous CTLs. Thus, Nef inhibitors represent an innovative approach to antiretroviral therapy that may provide a path to eradication of viral reser- voirs. Our most promising class of inhibitors (hydroxypyrazoles) bind tightly to their Nef protein target in vitro with KD values in the nM to pM range. However, Nef lacks an active site, which has complicated traditional medicinal chemistry optimization of existing compounds for in vivo testing due to the lack of correlation between Nef binding affinity in vitro and antiretroviral activity in cell-based systems. To circumvent this issue, we propose to use our existing Nef-binding compounds to develop Proteolytic Targeting Chimera (PROTAC) molecules for the targeted destruction of the Nef protein in HIV-infected cells. In this approach, existing hydroxypyrazole Nef-binding compounds will be coupled to ligands for ubiquitin E3 lig- ases via a flexible linker. The resulting Nef PROTACs are anticipated to catalyze the proteolytic degradation of Nef via E3-mediated polyubiquitination and proteasomal targeting. A major advantage of the PROTAC approach is that it requires only a selective binder of the target protein (Nef in this case) and not a functional inhibitor. We anticipate that selective PROTAC-mediated degradation will eliminate all Nef functions, including rescue of cell surface MHC-I mediated HIV-1 antigen presentation, leading to clearance of HIV+ cells via the CTL response as part of a strategy for latent reservoir reduction and functional cure. More generally, the PROTAC approach has generated a great deal of excitement in the pharmaceutical industry, because it provides new avenues to inhibit proteins previously considered undruggable. While PROTACs have higher molecular weights that typical small molecule drugs, recent preclinical and Phase I clinical studies have demonstrated activity in vivo as well as oral bioavailability. For this Phase I STTR project, HIV-1 Nef PROTAC development will combine the pharmaceutical and medicinal chemistry expertise of the Fox Chase Chemical Diversity Center, Inc. (www.fc-cdci.com; FCCDC) with the expertise of the Smithgall Lab at the University of Pittsburgh in HIV-1 Nef structure, function and inhibitor analysis. Our broad goal is to synthesize and test a series of Nef PROTACs with different Nef-targeting moieties, linkers, and E3 ubiquitin ligase ligands to identify compounds suitable for in vivo proof-of-concept studies. Suc- cessful completion of Phase I will provide a strong foundation for an expanded drug development program in Phase II, with the ultimate goal of clinical translation.